Molecular detection of Babesia and Theileria from crossbred cattle in Sirajganj and Rangpur districts of Bangladesh

Abstract Background Babesia and Theileria are potential threats to the livestock industry, causing considerable economic losses. These tick‐borne blood parasites are more prevalent in crossbred cattle than local cattle in Bangladesh. Objectives To confirm the species of Babesia and Theileria in crossbred cattle from the northern part of Bangladesh using conventional and molecular tools. Methods A total of 385 crossbred cattle blood samples were subjected to DNA extraction and PCR. For molecular detection, B. bigemina rhoptry‐associated protein 1a, B. bovis spherical body protein‐4, and Theileria spp. 18S rRNA were used as the marker genes. Results Using PCR, only 72 (18.7%) samples were found piroplasm positive, of which 12.2% Theileria, 4.7% Babesia, and 1.8% mixed infections. Both Babesia (7.3%), Theileria (7.7%) and mixed (2.8%) infections were detected in Sirajganj, and only Theileria (20.4%) was detected in Rangpur district. By PCR and nPCR we detected B. bigemina and T. annulata in Sirajganj district, and Theileria sp. in Rangpur district. The target gene sequences of isolated pathogens confirmed B. bigemina and T. annulata, and Theileria sp from these samples. Blood smears of all samples were also examined microscopically for Babesia and/or Theileria spp. and 14.3% of samples were found positive, of which 5.9% Babesia and 8.3% Theileria. Generally, the pathogens detected in Sirajgang and Rangpur were genetically related to South Asia, particularly South East Asian isolates. Conclusions These findings provide information for a better understanding of the epidemiology of Babesia and Theileria as well as to improve the approaches for diagnosis and control of tick‐borne diseases in Bangladesh.


INTRODUCTION
Babesiosis and theileriosis cause significant economic losses in the tropics and subtropics (Inci et al., 2007;Uilenberg, 1995). The economic losses are due to loss of body weight, milk output and animal death, as well as indirectly for the expenses related to therapeutic management of clinical cases and targeting control campaigns (Guswanto et al., 2017). In Bangladesh, babesiosis (2.3-9.0%) and theileriosis (5.8-8.5%) are found in both local and crossbred cattle (Al Mahmud et al., 2015;Alim et al., 2012;Hosen et al., 2020;Roy et al., 2018;Samad et al., 1983). Crossbred cattle had a greater prevalence of bovine piroplasmosis than local cattle (Siddiki et al., 2010). These tick-borne blood parasites are prevalent in crossbred cattle rearing in Sirajganj and Rangpur districts   (Ghosh et al., 2007;Kabir et al., 2011;Shuvo et al., 2021). However, the geo-climatic conditions of Bangladesh are thought to be very conducive to a wide variety of parasites, including ticks (Dey et al., 2020). The presence of a variety of tick species enhances the transmission of tick-borne diseases, especially babesiosis and theileriosis.
The diagnosis of these important blood protozoa are mainly based on clinical symptoms and microscopic examination of Giemsa-stained blood smears in Bangladesh. Several researchers identified bovine piroplasms using microscopic examination for epidemiological investigation (Al Mahmud et al., 2015;Chowdhury, Hossain et al., 2006).
Limited studies also have performed serological examination to confirm the tentative diagnosis of infections (Ali et al., 2016;Rahman, Sumon et al., 2015). However, these studies are only reliable for the diagnosis of acute cases but have limited value in subclinical cases due to low parasitaemia (Alim et al., 2012;Hosen et al., 2020;Samad et al., 1983). Compared to serological testing and microscopic identification of piroplasms, molecular detection of piroplasms infection by polymerase chain reaction (PCR) has proven to be very sensitive, accurate, and the gold standard test particularly in detecting Babesia and Theileria in carrier cattle (Belotindos et al., 2014).
B. bigemina rhoptry-associated protein 1a (RAP-1a) is a highly conserved gene among B. bigemina isolates that has been utilised in several studies (Guswanto et al., 2017;Ibrahim et al., 2013;Moumouni et al., 2015;Terkawi et al., 2012). The B. bovis spherical body protein-4 (SBP-4) gene is conserved between geographical isolates and has little homology with other apicomplexan parasites, emphasising the gene's usefulness as a particular target for molecular diagnosis (de Vries et al., 2006;Ruef et al., 2000). On the other hand, the 18S rRNA gene has been widely used for the identification and phylogenies of Theileria (Bawm et al., 2021;Mohamed et al., 2018).
In recent years, molecular studies have been performed to detect piroplasmosis in cattle of Mymensingh (Roy et al., 2018)  The selected areas were Kaunia, Pirgacha and Rangpur Sadar upazilas ( Figure 1).

Sample collection
Blood samples were collected from randomly selected 385 crossbred (local breed crossed with Holstein-Friesian and Sahiwal) cattle from 80 different dairy farms. Of these samples, 248 were from Sirajganj and 137 from Rangpur district, Bangladesh. The cattle were reared in semi-intensive and free-range systems (bathan). Blood samples were collected by ear puncture in 4 ml EDTA-coated tubes. A few drops of blood were immediately used to prepare thin blood smears, air dried, fixed in methyl alcohol for 2 min and brought to the laboratory for further processing. The remaining samples were kept at -20 • C until DNA extraction.

Microscopic examination
Thin blood smears were prepared, fixed with methanol, and stained with 10% Giemsa stain for 30 min. After staining, slides were washed with tap water and examined at 100× objective magnification by adding immersion oil. Each sample was examined in triplicate.

DNA extraction
Genomic

Molecular detection of Babesia and Theileria spp
To confirm the Babesia, PCR was conducted targeting the amplification of rhoptry-associated protein-1a (RAP-1a) gene of B. bigemina and spherical body protein-4 (SBP-4) of B. bovis through PCR and nPCR using species-specific primers (Table 1). To detect B. bigemina, PCR was performed with the following cyclic conditions: 95 • C for 5 min, 35 cycles at 94 • C for 1 min, 55 • C for 1 min, 72 • C for 1 min, and a final extension of 72 • C for 10 min (Terkawi et al., 2011). Similar conditions were used in nPCR to amplify the 412 bp region of RAP-1a gene of B. bigemina . For the detection of B. bovis, PCR conditions were as follows: initial denatu-ration at 94 • C for 1 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 65 • C for 1 min, extension at 72 • C for 1 min, and a final extension at 72 • C for 7 min. The conditions for nPCR were similar except for the annealing temperature, which was 53.5 • C (Terkawi et al., 2011).
Theileria species were identified through the amplification of the 18S rRNA gene. The reaction conditions were 95 • C for 5 min, 30 cycles at 95 • C for 30 s, 54 • C for 30 s, 72 • C for 1 min, and a final extension of 72 • C for 9 min (Wamuyu et al., 2015).
Double distilled water was used as a negative control for all PCR. The PCR was done in an Applied Biosystems TM 2720 thermal cycler. The amplified product of PCR assay was analysed by gel

2.6
Sequencing of DNA

Phylogenetic analysis
The sequences of ITS-1 were aligned using the program Clustal W within MEGA v.6.0 (Tamura et al., 2011). The sequence results were compared with the available sequence in the GenBank database using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm. nih.gov/Blast.cgi). The phylogenetic trees were constructed using the neighbour-joining method in MEGA version 6.0 (Tamura et al., 2013).
Bootstrap analysis with 1000 replications was used to estimate the confidence levels of the branching patterns of the trees. Representative sequences that were identified in this study were deposited in the

Microscopic detection of Babesia and Theileria
A total of 385 blood samples (248 samples from Sirajganj and 137 samples from Rangpur district) were collected and microscopically examined to detect Babesia or Theileria (Figure 2 Table 2.

Blast analysis and sequence alignment
The

Phylogenetic analysis
The The present study investigated the samples from crossbred cattle rearing in the selected farms or bathan, which provided clear data for understanding the epidemiology of tick-borne diseases in Bangladesh.
The detection rates of Babesia and Theileria were higher in PCR Since babesiosis and theileriosis are associated with the availability of ticks, therefore, routine diagnostic procedures, isolation of carrier animals from herds, and best management practices with clean environment are necessary for controlling these diseases in Bangladesh.

CONCLUSION
In the present study, 14.3% crossbred cattle were infected with Babesia

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The authors tried to maintain the highest possible ethical standards in their works without any injury of animals. The study was approved by the Animal Welfare and Experimentation Ethical Committee of Bangladesh Agricultural University. The approval number is AWEEC/BAU/2017 (16).

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.989